Research ArticleHEALTH AND MEDICINE

Mechanical deformation induces depolarization of neutrophils

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Science Advances  14 Jun 2017:
Vol. 3, no. 6, e1602536
DOI: 10.1126/sciadv.1602536
  • Fig. 1 Morphological changes in activated neutrophils.

    (A) OS with two counterpropagating laser beams emanating from single-mode optical fibers, enabling contactless morphometry. (B) Confocal images of Syto 61 dye– and MitoTracker Orange–stained neutrophils. Resting cells remain round (top), whereas activated cells form amoeboid shapes (bottom). (C) Phase contrast images of cells trapped in the OS with strong white halo simplifying contour detection. (D) Morphometry using phase contrast images from (C). Edge detection algorithms are used to fit ellipses (red line) to the contours of the cells (cyan line), giving access to the semimajor and semiminor axes, a and b, respectively. (E) Plots of the aspect ratio, a/b, from the morphometry results of (D). Activated neutrophils have a significantly higher aspect ratio compared to resting neutrophils. (F) Mean circularity of F-actin cortex of cells stained with Alexa Fluor 488 Phalloidin. After fMLP and GM-CSF treatment, cellular circularity decreases significantly. ***P < 0.001 (significant difference). Scale bars, 5 μm. Both GM-CSF (10 ng/ml)– and fMLP (100 nM)–treated neutrophils have significantly (P < 0.0001) higher CD11b expression (fig. S1) and lower CD62L expression (fig. S2) compared to resting neutrophils (for details of priming, see the Supplementary Materials). In addition, intracellular ROS production was enhanced in primed neutrophils (fig. S3).

  • Fig. 2 Reduced deformability of primed neutrophils in the OS.

    (A) Representative strain and compliance time course (mean ± SEM) from n ≥ 4 experiments for resting (n = 56), fMLP-treated (n = 24), and GM-CSF–treated (n =39) neutrophils. Reduction in strain and compliance is seen in GM-CSF– and fMLP-treated cells. (B) fMLP- and GM-CSF–treated neutrophils are significantly stiffer than resting cells [****P < 0.0001 and ***P < 0.001 (significant difference)]. (C) Confocal images of Alexa Fluor 488 Phalloidin–stained cells showing F-actin distribution in fixed resting and chemically activated neutrophils. Scale bar, 5 μm.

  • Fig. 3 Mechanically induced priming and depolarization of neutrophils.

    (A) Multiple stretching of round, resting neutrophils in the OS using sinusoidal pulses of laser power (yellow) leads to stiffening of about 40% of the cells (red; n = 11) within 20 to 30 s, acquiring the primed morphology established in Fig. 1 (see inset picture at t = 50 s), compared to still resting cells (black; n = 14). Representative example of n = 3 experiments. (B) Bar graph of average strain of the two groups in (A) at t = 50 s. (C) Repeated sinusoidal pulses of laser power (Pmax = 1 W per fiber; yellow) cause fMLP-primed neutrophils (inset picture of typical cell at t = 0 s) to depolarize, as evidenced by the retraction of pseudopods and decrease in aspect ratio (inset picture, right) (see also movies S2 and S3). The temporal evolution of the average aspect ratio of n = 28 cells, which is typical for n = 4 repeats, is shown. (D) Box plots of the average aspect ratio of fMLP-treated cells in (C) at t = 0 s and t = 50 s. *P < 0.05 and ***P < 0.001 (significant difference). Scale bars, 5 μm.

  • Fig. 4 Delayed transit of activated cells in the MMM.

    (A) Schematic of the MMM, illustrating constrictions and inlet and outlet channels from and to reservoirs (not shown). (B) Actual micrograph of MMM showing all 187 constrictions. The minimum gaps at the constrictions are smaller than the diameter of cells, ensuring that each cell is repeatedly deformed during the advection. (C) Box plots of average transit times at 50-mbar driving pressure for various cell states (n = 50 per state), showing a statistically significant increase in transit times for all activated cells, compared to resting neutrophils. (D) Bar graph of average (error bar is SEM) transit times of resting, fMLP-, and GM-CSF–treated neutrophils at 37°C and pressures of 10, 50, and 100 mbar. Note that, at 10 mbar, the GM-CSF–treated cells could hardly make it through the device; hence, there are no data for this cell state at 10 mbar. **P < 0.01 and ****P < 0.0001 (significant difference). Scale bars, 15 μm.

  • Fig. 5 Depolarization in the MMM: shape change and molecular readouts.

    (A) Chemically primed neutrophils at the inlet showing pseudopods, round up in the outlet within 5 min following advection through constrictions. Images are representative of n = 5 experiments at 50-mbar driving pressure with MMM minimum gaps of 5 μm. (B) Box plot showing the quantification of shape changes (A), with a significant (P < 0.05) increase in circularity following stenotic advection for cells in the outlet, compared to cells in the inlet. (C) Confocal images of fMLP-treated CD11b+ neutrophils before (inlet) and 5 min after (outlet) stenotic advection in MMM at 50 mbar (minimum gap of 5 μm). (D) Box plots of CD11b intensity from (C) showing a significant reduction (P < 0.05) in CD11b intensity about 5 min following stenotic advection. Scale bars, 50 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1602536/DC1

    Supplementary Materials and Methods

    fig. S1. Chemical priming of resting neutrophils leads to increase in CD11b expression.

    fig. S2. Chemical priming of resting neutrophils leads to decrease in CD62L expression.

    fig. S3. Higher ROS production in activated neutrophils compared to resting neutrophils.

    fig. S4. Average aspect ratio during mechanical priming of neutrophils.

    fig. S5. Mechanically induced depolarization of a chemically primed neutrophil.

    fig. S6. Mechanically induced depolarization of GM-CSF–primed neutrophils.

    fig. S7. Mechanically induced depolarization of a chemically primed neutrophil at a lower laser power.

    fig. S8. Mechanically induced priming and depolarization of resting neutrophils.

    fig. S9. Viability tests using trypan blue and annexin V/propidium iodide.

    fig. S10. Delayed transit of activated cells in MMM at a lower temperature of 24°C.

    movie S1. Mechanical deformation causes priming of resting neutrophils.

    movie S2. Mechanically induced depolarization of PMN (fMLP-treated).

    movie S3. Mechanically induced depolarization of PMN (GM-CSF–treated).

    movie S4. Mechanically induced depolarization of PMN in OS without thermal effects.

    movie S5. RvE1-induced recircularization of mechanically polarized PMN.

    movie S6. Confirmation of OS results with MMM.

    movie S7. Activation of some resting cells in MMM constrictions.

    Other materials S1 to S3. Consent forms and questionnaire for blood donors.

    References (5659)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Chemical priming of resting neutrophils leads to increase in CD11b expression.
    • fig. S2. Chemical priming of resting neutrophils leads to decrease in CD62L expression.
    • fig. S3. Higher ROS production in activated neutrophils compared to resting neutrophils.
    • fig. S4. Average aspect ratio during mechanical priming of neutrophils.
    • fig. S5. Mechanically induced depolarization of a chemically primed neutrophil.
    • fig. S6. Mechanically induced depolarization of GM-CSF–primed neutrophils.
    • fig. S7. Mechanically induced depolarization of a chemically primed neutrophil at a lower laser power.
    • fig. S8. Mechanically induced priming and depolarization of resting neutrophils.
    • fig. S9. Viability tests using trypan blue and annexin V/propidium iodide.
    • fig. S10. Delayed transit of activated cells in MMM at a lower temperature of 24°C.
    • Legends for movies S1 to S7
    • Other materials S1 to S3. Consent forms and questionnaire for blood donors.
    • References (56–59)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.wmv format). Mechanical deformation causes priming of resting neutrophils.
    • movie S2 (.wmv format). Mechanically induced depolarization of PMN (fMLP-treated).
    • movie S3 (.wmv format). Mechanically induced depolarization of PMN (GM-CSF–treated).
    • movie S4 (.wmv format). Mechanically induced depolarization of PMN in OS without thermal effects.
    • movie S5 (.avi format). RvE1-induced recircularization of mechanically polarized PMN.
    • movie S6 (.wmv format). Confirmation of OS results with MMM.
    • movie S7 (.wmv format). Activation of some resting cells in MMM constrictions.

    Download Movies S1 to S7

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